Inflammatory bowel diseases (IBD) are highly prevalent intestinal diseases in the United States affecting 1.4 million individuals. These diseases are associated with reduced quality of life and psychological co-morbidity. Current estimates for IBD associated treatment costs in the US are $6.3 billion. The high rate of recurrence and lack of safe and curative treatments for IBD underscore the need for alternate therapeutic approaches for these complex diseases. Bacterial proteases capable of inducing intestinal permeability via protease-activated receptors (PARs) present a novel therapeutic target for IBD. The goals for this proposal are to (I) elucidate the specific mechanism by which enteric bacteria induce intestinal permeability, (II) assess the biological effect of protease producing and inhibiting enteric bacteria on intestinal permeability, and (III) assess the inflammatory effect of protease producing and inhibiting enteric bacteria on the intestine. To address these goals we have proposed three specific aims. In specific aim 1 we will determine the ability of protease-producing and - inhibiting enteric bacterial strains to regulate PARs and permeability in human epithelial cells in vitro. To achieve this aim we will use wild-type and mutant Enterococcus faecalis OG1RF strains that lack gelatinase or serine protease activity, and wild-type and mutant Bifidobacterium longum ATCC15707 strains that lack serpin activity. We will expose tight junction forming T-84 epithelial cell monolayers to parental and mutant E. faecalis OG1RF and B. longum ATCC15707 strains and measure cell permeability and PAR activation. In specific aim 2 we will determine the ability of protease-producing and inhibiting enteric bacterial strains to regulate PAR- dependent intestinal permeability in gnotobiotic mice. To achieve this aim we will mono- and dual-associate germ-free wild-type and PAR deficient mice with parental and mutant bacterial strains and measure intestinal permeability and PAR activation. In specific aim 3 we will determine the ability of protease-producing and inhibiting enteric bacterial strains to induce intestinal permeability and inflammation in gnotobiotic IL-10 deficient (IL-10-/-) mice. To achieve this aim we will mono- and dual-associate germ-free wild-type and IL-10-/- mice with parental and mutant bacterial strains and measure intestinal permeability, PAR activation, and inflammation. The contribution of the proposed research is significant as it will define a precise mechanism by which enteric bacteria alter intestinal permeability and contribute to inflammation. The proposed research will also be of significance because the outcome will contribute to the broader understanding of mechanisms by which dysbiosis in the intestinal microbiota affects IBD. Additionally, the proposed research is innovative in our opinion as it will allow for the design of rational pathophysiology-directed probiotic treatment of GI disease. PUBLIC HEALTH RELEVANCE: The proposed project is relevant to public health as it will identify a novel therapeutic approach for Inflammatory Bowel Diseases (IBD) via modulation of the intestinal microbiota. The aims outlined in this proposal will (I) elucidate the specific mechanism by which enteric bacteria induce intestinal permeability, (II) investigate the biological effect of protease-producing and -inhibiting enteric bacteria on intestinal permeability, and (III) investigate the inflammatory effect of protease-producing and -inhibiting enteric bacteria on the intestine. This proposal will identify bacterial proteases and intestinal permeability as novel pathophysiology- directed therapeutic targets for IBD. Additionally, this proposal will identify serine protease inhibitor (serpin)- producing bacteria as probiotic agents for the management of IBD.